An HPLC-based assay for improved measurement of glutamate decarboxylase inhibition/activation

IF 4.4 3区 医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Marija S. Genčić , Nikola M. Stojanović , Marko Z. Mladenović , Niko S. Radulović
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引用次数: 2

Abstract

L-Glutamic acid decarboxylase (GAD) is an enzyme that ensures the balance between the levels of two neurotransmitters, γ-aminobutyric acid (GABA) and L-glutamic acid (L-Glu), necessary for proper brain functioning. A reduction in the concentrations of GABA and/or GAD activity has been implicated in the symptoms associated with epilepsy, which could be plausibly alleviated by the application of GAD activators. As any unnecessary interference in GAD catalytic activity could be detrimental, it is important to study whether CNS (or other) drug candidates act on GAD or not. The ability to identify and reduce this risk early could significantly improve the process of drug development. Although many methods for measuring GAD activity in various biological samples have been described, only few (such as manometric and radiometric) were adopted as in vitro assays for the screening of potential GAD inhibitors/activators. However, these methods require specialized equipment and/or an expensive radiolabeled substrate, and may have sensitivity and/or reliability issues. Therefore, this study aimed to develop an HPLC-DAD-based assay that would allow a simple and more accurate measurement of GAD inhibition or activation using unpurified mice or rat brain homogenates. This assay is based on the quantification of GABA, formed during the enzymatic reaction, after its derivatization with dansyl chloride. Various parameters were evaluated to optimize the assay procedure (e.g. homogenate volume, incubation time, DMSO content, GAD, GABA, and dansyl-GABA stabilities). This assay was validated for pharmacological screenings using 3-mercaptopropionic acid and gallic acid and GAD obtained from different experimental animals.

Abstract Image

谷氨酸脱羧酶抑制/激活的高效液相色谱检测方法
l -谷氨酸脱羧酶(GAD)是一种确保两种神经递质γ-氨基丁酸(GABA)和l -谷氨酸(L-Glu)水平平衡的酶,是正常大脑功能所必需的。GABA浓度和/或广泛性焦虑症活性的降低与癫痫相关症状有关,应用广泛性焦虑症激活剂似乎可以减轻这种症状。由于对GAD催化活性的任何不必要的干扰都可能是有害的,因此研究CNS(或其他)候选药物是否对GAD起作用是很重要的。早期识别和减少这种风险的能力可以显著改善药物开发过程。尽管已经描述了许多测量各种生物样品中GAD活性的方法,但只有少数方法(如压力测定法和辐射测定法)被用于筛选潜在的GAD抑制剂/活化剂的体外测定。然而,这些方法需要专门的设备和/或昂贵的放射性标记基板,并且可能存在灵敏度和/或可靠性问题。因此,本研究旨在开发一种基于hplc - dad的检测方法,使用未纯化的小鼠或大鼠脑匀浆,可以更简单、更准确地测量GAD抑制或激活。这种分析是基于定量的GABA,形成在酶促反应,与氯丹酰衍生后。评估各种参数以优化分析程序(例如匀浆体积,孵育时间,DMSO含量,GAD, GABA和丹酚-GABA稳定性)。利用从不同实验动物中获得的3-巯基丙酸、没食子酸和GAD进行药理筛选,验证了该方法的有效性。
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来源期刊
Neurochemistry international
Neurochemistry international 医学-神经科学
CiteScore
8.40
自引率
2.40%
发文量
128
审稿时长
37 days
期刊介绍: Neurochemistry International is devoted to the rapid publication of outstanding original articles and timely reviews in neurochemistry. Manuscripts on a broad range of topics will be considered, including molecular and cellular neurochemistry, neuropharmacology and genetic aspects of CNS function, neuroimmunology, metabolism as well as the neurochemistry of neurological and psychiatric disorders of the CNS.
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